Genome-wide analysis of p53-regulated transcription in Myc-driven lymphomas.

The tumour suppressor p53 is a transcription factor that controls cellular stress responses. Here, we dissected the transcriptional programmes triggered upon restoration of p53 in Myc-driven lymphomas, based on the integrated analysis of p53 genomic occupancy and gene regulation. p53 binding sites were identified at promoters and enhancers, both characterized by the pre-existence of active chromatin marks. Only a small fraction of these sites showed the 20 base-pair p53 consensus motif, suggesting that p53 recruitment to genomic DNA was primarily mediated through protein-protein interactions in a chromatin context. p53 also targeted distal sites devoid of activation marks, at which binding was prevalently driven by sequence recognition. In all instances, the relevant motif was the canonical unsplit consensus element, with no clear evidence for p53 recruitment by split motifs. At promoters, p53 binding to the consensus motif was associated with gene induction, but not repression, indicating that the latter was most likely indirect. Altogether, our data highlight key features of genome recognition by p53 and provide unprecedented insight into the pathways associated with p53 reactivation and tumour regression, paving the way for their therapeutic application.

Dual regulation of Myc by Abl.

The tyrosine kinase c-Abl (or Abl) and the prolyl-isomerase Pin1 cooperatively activate the transcription factor p73 by enhancing recruitment of the acetyltransferase p300. As the transcription factor c-Myc (or Myc) is a known target of Pin1 and p300, we hypothesized that it might be regulated in a similar manner. Consistent with this hypothesis, overexpression of Pin1 augmented the interaction of Myc with p300 and transcriptional activity. The action of Abl, however, was more complex than predicted. On one hand, Abl indirectly enhanced phosphorylation of Myc on Ser 62 and Thr 58, its association with Pin1 and p300 and its acetylation by p300. These effects of Abl were exerted through phosphorylation of substrate(s) other than Myc itself. On the other hand, Abl interacted with the C-terminal domain of Myc and phosphorylated up to five tyrosine residues in its N-terminus, the principal of which was Y74. Indirect immunofluorescence or immunohistochemical staining suggested that the Y74-phosphorylated form of Myc (Myc-pY74) localized to the cytoplasm and coexisted either with active Abl in a subset of mammary carcinomas or with Bcr-Abl in chronic myeloid leukemia. In all instances, Myc-pY74 constituted a minor fraction of the cellular Myc protein. Thus, our data unravel two potential effects of Abl on Myc: first, Abl signaling can indirectly augment acetylation of Myc by p300, and most likely also its transcriptional activity in the nucleus; second, Abl can directly phosphorylate Myc on tyrosine: the resulting form of Myc appears to be cytoplasmic, and its presence correlates with Abl activation in cancer.

Genome-wide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts.

The transition from quiescence to proliferation is a key regulatory step that can be induced by serum stimulation in cultured fibroblasts. The transcription factor Myc is directly induced by serum mitogens and drives a secondary gene expression program that remains largely unknown. Using mRNA profiling, we identify close to 300 Myc-dependent serum response (MDSR) genes, which are induced by serum in a Myc-dependent manner in mouse fibroblasts. Mapping of genomic Myc-binding sites by ChIP-seq technology revealed that most MDSR genes were directly targeted by Myc, but represented a minor fraction (5.5%) of all Myc-bound promoters (which were 22.4% of all promoters). Other target loci were either induced by serum in a Myc-independent manner, were not significantly regulated or were negatively regulated. MDSR gene products were involved in a variety of processes, including nucleotide biosynthesis, ribosome biogenesis, DNA replication and RNA control. Of the 29 MDSR genes targeted by RNA interference, three showed a requirement for cell-cycle entry upon serum stimulation and 11 for long-term proliferation and/or survival. Hence, proper coordination of key regulatory and biosynthetic pathways following mitogenic stimulation relies upon the concerted regulation of multiple Myc-dependent genes.

A positive role for Myc in TGFbeta-induced Snail transcription and epithelial-to-mesenchymal transition.

Myc and transforming growth factor-beta (TGFbeta) signaling are mutually antagonistic, that is Myc suppresses the activation of TGFbeta-induced genes, whereas TGFbeta represses c-myc transcription. Here, we report a positive role for Myc in the TGFbeta response, consisting in the induction of an epithelial-to-mesenchymal transition (EMT) and the activation of the EMT-associated gene Snail. Knockdown of either Myc or the TGFbeta effectors SMAD3/4 in epithelial cells eliminated Snail induction by TGFbeta. Both Myc and SMAD complexes targeted the Snail promoter in vivo, DNA binding occurring in a mutually independent manner. Myc was bound prior to TGFbeta treatment, and was required for rapid Snail activation upon SMAD binding induced by TGFbeta. On the other hand, c-myc downregulation by TGFbeta was a slower event, occurring after Snail induction. The response of Snail to another cytokine, hepatocyte growth factor (HGF), also depended on Myc and SMAD4. Thus, contrary to their antagonistic effects on Cip1 and INK4b, Myc and SMADs cooperate in signal-dependent activation of Snail in epithelial cells. Although Myc also targeted the Snail promoter in serum-stimulated fibroblasts, it was dispensable for its activation in these conditions, further illustrating that the action of Myc in transcriptional regulation is context-dependent. Our findings suggest that Myc and TGFbeta signaling may cooperate in promoting EMT and metastasis in carcinomas.

Translation of a retained intron in tyrosinase-related protein (TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage.

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

IFN-gamma gene transfer restores HLA-class I expression and MAGE-3 antigen presentation to CTL in HLA-deficient small cell lung cancer.

In this study, we have analyzed the possibility of inducing T cell responses against small cell lung cancer (SCLC), a still incurable tumor, by cytokine gene transfer approaches. By RT-PCR analysis most SCLC expressed the CTL-defined tumor antigens MAGE-3 (10/11), MAGE-1 (7/11) and less frequently BAGE (4/11) and GAGE1,2 (4/11). Although the surface expression of HLA class I molecules was low on most SCLC, thus preventing CTL recognition, treatment of the cells with IFN-gamma enhanced HLA-class I levels in all cases. Two MAGE3+ SCLC cell lines displaying the A2 HLA-class I allele, involved in MAGE-3 antigen presentation to CTL, were stably transfected with the IFN-gamma gene (alone or co-transfected with IL-2). IFN-gamma-transfected cells displayed a clearcut increase in expression of HLA-class I and beta 2-microglobulin at both protein and mRNA level, and of TAP-1 and TAP-2 mRNA. Perhaps more importantly, IFN-gamma transfected cells were recognized by the MAGE-3-specific A2-restricted antimelanoma CTL clone 297/22, while unmodified cells or cells transfected with the IL-2 gene alone were not. These data indicate that IFN-gamma gene transfection into HLA-deficient SCLC cells is able to restore their ability to present endogenous tumor antigens to CTL and that IFN-gamma gene transfer approaches may be attempted to induce specific CTL responses in SCLC.

Expression of the MAGE gene family in primary and metastatic human breast cancer: implications for tumor antigen-specific immunotherapy.

Some human tumors express known antigens that can be utilized as targets for specific immunotherapy. An absolute requirement for the efficacy of this therapeutic strategy is an adequate expression of the candidate antigen by all cells of the primary and metastatic tumor. To examine the presence and distribution of tumor-associated antigens in metastatic breast cancer, we used PCR analysis and ethidium bromide staining to test the expression of genes of the MAGE family in 28 primary tumors and related metastatic samples. Overall, samples obtained from 7 of 28 patients revealed positive. However, 2 of 3 primary tumors positive for MAGE-1 and/or MAGE-3 had corresponding negative metastatic lesions. On the contrary, 4 of the 25 MAGE-negative primary tumors gave rise to positive metastatic nodes. Our results confirm in vivo, at the molecular level, the tumor-antigen heterogeneity previously observed at the cellular level by in vitro analysis. Our data strongly suggest that, at least in patients with breast cancer, multiple different antigens would be required to optimize the recognition of neoplastic cells in immunotherapeutic protocols using MAGE products as target antigens.

Insulin-mimetic effects of vanadate in preventing the increase of P450IIIA and P450IA subfamily proteins in streptozotocin-diabetic rats.

The insulin-mimetic effects of vanadate in preventing the increase in the level and activity of several P450 proteins in streptozotocin-diabetic rats were examined, in order to extend knowledge of the insulin-like actions of vanadate from glucose metabolism to P450-dependent metabolism. The diabetic state caused by the pancreatic beta cell toxin streptozotocin results, like the diabetes of genetic origin, in major alterations in the expression of P450 isozymes. We focused our attention on the P450III and P450I isoforms, known to be altered during the onset of diabetes. We found an increase in P450IIIA1-linked erythromycin demethylase activity (to about double the control level) and in the relative levels of P450III and P450I isozymes after 2 weeks of uncontrolled diabetes. These parameters were not different from control values in rats given vanadate in drinking water for 2 weeks after streptozotocin administration or in insulin-treated rats. In summary, vanadate appears to exert insulin-mimetic actions on the P450III and P450I family proteins that have a key role in cytochrome P450-dependent metabolism.